Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4

Sall4 encodes a transcription factor and is known to participate in the pluripotency network of embryonic stem cells. Sall4 expression is known to be high in early stage post-implantation mouse embryos. During early post-gastrulation stages, Sall4 is highly expressed in the tail bud and distal limb buds, where progenitor cells are maintained in an undifferentiated status. The expression of Sall4 is rapidly downregulated during embryonic development. We previously demonstrated that Sall4 is required for limb and posterior axial skeleton development by conditional deletion of Sall4 in the T (Brachyury) lineage. To gain insight into Sall4 functions in embryonic development and postnatal digit regeneration, we genetically overexpressed Sall4 in the mesodermal lineage by the TCre transgene and a novel knockin allele of Rosa26-loxP-stop-loxP-Sall4. In significant contrast to severe defects by Sall4 loss of function reported in previous studies, overexpression of Sall4 resulted in normal morphology and pattern in embryos and neonates. The length of limb long bones showed subtle reduction in Sall4-overexpression mice. It is known that the digit tip of neonatal mice has level-specific regenerative ability after experimental amputation. We observed Sall4 expression in the digit tip by using a sensitive Sall4-LacZ knock-in reporter expression. Sall4 overexpression did not alter the regenerative ability of the terminal phalange that normally regenerates after amputation. Moreover, Sall4 overexpression did not confer regenerative ability to the second phalange that normally does not regenerate after amputation. These genetic experiments show that overexpression of Sall4 does not alter the development of the appendicular and axial skeleton, or neonatal digit regeneration. The results suggest that Sall4 acts as a permissive factor rather than playing an instructive role.


Introduction
Sall4 encodes a zinc finger transcription factor [1,2], and mutations in human SALL4 cause Duane-radial ray syndrome, an autosomal dominant disorder [3,4]. Patients  remain largely unknown in post-implantation mouse embryos due to the early lethality of Sall4 null mutants [6][7][8][9]. To gain insight into Sall4 functions, we genetically overexpressed Sall4 by recombination using the TCre transgene. In significant contrast to severe defects in reported Sall4 loss of function studies, our results show that Sall4 overexpression in the T-lineage results in the normal development of mouse embryos and no alteration of digit tip regenerative ability.

Generation of the inducible Sall4 knock-in allele and mice breeding
The inducible Sall4 expression vector was generated as previously described [33] with replacement of Dmrt1 by the Flag-Sall4a-T2A-HA-Sall4b sequence. The expression cassette was knocked into the Rosa26 locus by homologous recombination in R1 mouse ES cells, as diagrammed in Fig 1. The correctly targeted ES cells were injected into C57BL/6 blastocysts after confirming the normal karyotype, and chimeras were generated. Germline transmission was confirmed by breeding chimeras with C57BL/6 mice and genomic PCR of offspring, and the mouse line was termed as R26-Sall4. The R26-Sall4 mice were bred with C57BL/6 mice at least 3 generations before experimentation. C57BL/6 and CD-1 mice were obtained from the Jackson Laboratory and Charles River, respectively. TCre mice were previously described [34] and backcrossed >12 generations with C57BL/6. For embryo experiments, R26-Sall4 mice were timed mated with TCre mice. For postnatal skeletal analysis and digit regeneration experiments, R26-Sall4 mice were bred with TCre mice. Experimental protocols were approved by the University of Minnesota Animal Care and Use Committee (protocol number: 1706-34894A, 1910-37482A).

ES cell screening and mouse genotyping
To screen targeted ES cells, a DNA fragment described in [33] was labelled using DIG DNA labeling mix (Roche, Cat#11277065910) and genomic Southern hybridization was performed following a standard procedure [35]. Genomic PCR of ES cell DNA and PCR genotyping on tail clip DNA for the R26-Sall4 mice were conducted as previously described [33].

Skeletal preparation and whole mount in situ hybridization
For alcian blue/alizarin red staining, neonates or the postnatal day (P) 21 autopod were fixed in 50% ethanol. Skeletal staining and whole mount in situ hybridization of embryos were performed as previously described [15,21].

Bone length measurement and statistical analysis
The length of the humerus and femur was measured by using FIJI. Lines were drawn parallel to the long axis of each bone, then perpendicular to those lines such that the lines touched the edges of the bone. The length of the bone was measured by using the distance between the edges of the line parallel to the long axis. The measurement was performed by two independent individuals, and the average value was used for statistical examination, which was performed by One-way ANOVA using JMP software version 16.

Neonatal digit amputation
Neonatal (P0) mice were anesthetized on ice and the digits of the hindlimbs were amputated [36]. The regenerative amputations were performed using microdissection scissors (Fine Science Tools, Cat#15000-00) cross the distal third of the terminal phalange [27]. For nonregenerative amputation, the distal third of the second phalange was amputated.

Western blotting
For embryos, E10.5 embryos were dissected, and EGFP-positive (TCre +/tg ; R26-Sall4 +/tg ) or negative (TCre +/+ ; R26-Sall4 +/tg ) embryos were grouped. Embryos were cut at the anterior to the forelimb buds, and the body was used for protein extraction using RIPA buffer with a The targeting vector also contains a diphtheria toxin negative selection cassette (PGK-DTA-bGH-PA) with diphtheria toxin expressed from the PGK promoter and followed by a bovine growth hormone poly-adenylation site. Homologous recombination into the Rosa26 locus removed PGK-DTA-bGH-PA and produced the single copy transgene CAG-Stop-Sall4-IRES-EGFP. Cre-mediated recombination deletes the "floxed" stop cassette to generate CAG-Sall4-IRES-EGFP. (C) Correct homologous recombination of the 5' homology arm of the targeting vector into Rosa26 was confirmed by PCR of ES cell genomic DNA using primers F1 and R1 (indicated in panel B), which amplify between exon 1 of Rosa26 and the CAG promoter. No amplification product was detected in wild-type ES cells, but the predicted 1248 bp amplicon was detected in a targeted ES cell clone (#4). Control DNA was prepared from the tail clip of conditional Dmrt1 expression mouse that was generated using the same knockin system (33). (D) Homologous recombination of the 3' vector arm was confirmed by Southern blotting using the 3' probe indicated in panel B. Wild-type ES cell DNA generated in detection of the predicted 11 kb fragment, whereas targeted clones (two right lanes) generated both the 11 kb fragment and a 9 kb fragment corresponding to the targeted allele. protease inhibitor cocktail (Thermo, Cat# 1862209). The extract was centrifuged at 14,000g for 10 min at 4˚C, and the supernatant was used for Western blotting.
For neonatal digits, the entire digits were combined, protein was extracted similar to the E10.5 embryos, and the lysate was subjected to Western blotting.

Generation of a recombination-dependent Sall4 overexpression mouse line
In order to test whether continued expression of Sall4 interferes with normal embryonic development, we sought to express Sall4 by Cre-dependent genetic recombination. We knocked in a Sall4 cassette into the Rosa26 locus as previously described [33]. The Sall4 transgene expression is silenced by the stop sequence in front of the Sall4 transgene, which is flanked by the loxP sequences. By Cre-dependent deletion of the stop sequence, Sall4 is expressed under a strong CAG promoter (Fig 1A and 1B). There are two Sall4 isoforms, Sall4a and Sall4b, which are generated through an internal splicing in exon 2 [37]. A previous study in mouse ES cells provided evidence that SALL4A and SALL4B have overlapping but non-identical binding sites [37]. Because functional differences between SALL4A and SALL4B during mouse development are unknown, we expressed both Sall4a and Sall4b. We inserted the Sall4a-T2A peptide sequence-Sall4b in the expression cassette, followed by the internal ribosome entry sequence and the EGFP sequence. Cre-dependent recombination causes expression of both Sall4a and Sall4b, as well as an EGFP reporter. This expression cassette is knocked into the Rosa26 locus in the R1 mouse ES cells [35]. After electroporation, the ES cells were selected by G418 treatment. Among the 64 clones isolated after G418 selection, six properly targeted embryonic stem cell colonies were confirmed by genomic Southern blotting and genomic PCR (Fig 1C  and 1D). After confirming normal karyotype, chimeric mice were generated by injecting one ES cell clone into blastocysts, which were bred with C57BL/6 mice. Germline transmission was confirmed by genomic PCR of offspring. This study was performed using this one strain.

Sall4 overexpression in the Brachyury lineage results in development of normal skeletal pattern
Sall4 expression is rapidly downregulated in post-gastrulating stages, and the high level of expression is confined in the distal limb buds and the tail buds [13,14]. Sall4 deletion in the Brachyury lineage using TCre caused severe defects in the appendicular and posterior axial skeleton [15,21]. In order to test whether continued expression of Sall4 affects those skeletons, we overexpressed Sall4 in the Brachyury lineage by crossing R26-Sall4 mice with TCre mice.
At E9.5, strong Sall4 expression is detected in the caudal part of the body and the distal part of the forelimb bud in control embryos (Fig 2A-2C, n = 3). In TCre; R26-Sall4 embryos, strong Sall4 expression was broadly detected in the trunk mesoderm, the entire forelimb buds and in the caudal part of the body (Fig 2D-2F, n = 3). The tissue with high levels of Sall4 expression correlates with the tissue with the EGFP signal that reports recombination and overexpression of the transgenes (Fig 2G and 2H, n = 3). Western blotting with an extract from the embryo (posterior to the anterior-edge of the forelimb bud) also showed an increased level of SALL4 in TCre +/tg ; R26-Sall4 +/tg , compared with TCre +/+ ; R26-Sall4 +/tg embryos. Specifically, the data show that SALL4A overexpression is greater than SALL4B overexpression, while SALL4B expression is increased from baseline in the control. The ratio of SALL4A/GAPDH signal intensity was increased 1.7-fold in TCre +/Tg ; R26-Sall4 +/tg embryos, compared with TCre +/+ ; R26-Sall4 +/tg embryos. Taken together, these results show that Sall4 is overexpressed by TCredependent recombination.

Sall4 is expressed in the digit tip
Next, we sought to determine whether Sall4 is expressed in the digit tip, which regenerates after experimental amputation [27]. Because endogenous Sall4 expression is rapidly downregulated in developing embryos and becomes difficult to detect, we made use of a sensitive LacZ reporter, knocked into the Sall4 locus [6]. By whole mount LacZ staining, we observed strong Sall4-LacZ reporter expression in the tail tip and genital organs at E14.5 (blue arrowhead and red arrowhead, respectively, Fig 4A, n = 4), as previously reported [14]. We also observed signals at the digit tip (black arrows, Fig 4A). The staining at the digit tip was observed at E16.5 (Fig 4B, n = 3, Fig 4D, n = 3) and in neonates (Fig 4C, n = 3, Fig 4E, n = 3). These data show Sall4 expression in the digit tip during late gestation to neonatal stages.

Sall4 overexpression does not alter the regenerative ability of the digit tip
The neonatal mouse digit regenerates after amputation at the terminal phalange level. In contrast, the digit fails to regenerate after amputation at the second phalange level [27]. We sought to investigate whether overexpression of Sall4 can alter regenerative ability of the digit tip. We first confirmed overexpression of SALL4 in the digit of TCre +/tg ; R26-Sall4 +/tg neonates with Western blotting. Tissue lysate prepared from the entire digits of TCre +/+ ; R26-Sall4 +/tg neonates did not show detectable SALL4. In contrast, digit lysate from TCre +/tg ; R26-Sall4 +/tg neonates showed overexpressed SALL4A and SALL4B (Fig 5A). We amputated the digit tip at the terminal phalange of control (TCre +/Tg ; R26-Sall4 +/+ , n = 7) and Sall4-overexpression (TCre +/  R26-Sall4 +/tg , n = 9) neonates, and stained the digits with alcian blue/alizarin red at day 21. The regenerated digit tip skeleton was comparable between the two groups (Fig 5B and 5C). This result indicates that overexpression of Sall4 did not impair regenerative ability of the digit tip. Next, we amputated at the digit at the second phalange level at the neonate stage. In both control (n = 7) and Sall4-overexpression (n = 9) mice, alcian blue/alizarin red staining showed no regeneration of the amputated digits (Fig 5D and 5E). This result indicates that overexpression of Sall4 does not supply regenerative ability to non-regenerative digits. Overall, these results indicate that Sall4 overexpression does not change regenerative and non-regenerative ability of the neonatal mouse digit tip.
Sall4 overexpression approach was also conducted to gain insight into Sall4 function in animal development. For example, full length Sall4 overexpression by plasmid electroporation into the head ectoderm in chick embryos showed that Sall4 can induce invagination of the non-placodal head ectoderm [46]. In this system, a region of interest is targeted by plasmid injection and electroporation, and the expression is lost as cells divide and dilute the electroporated plasmid. In another approach, synthetic sall4 mRNA was injected into blastomeres at the early cleavage stages of Xenopus [45]. With this approach, one can overexpress sall4 in the entire embryos or target specific blastomeres to spatially restrict overexpression. Nonetheless, as cells divide and the injected mRNA is degraded, the overexpressed transcripts are lost over the course of development. In our study, we used Cre-loxP-dependent conditional overexpression of Sall4 in mouse embryos. A Sall4 expression cassette is knocked into the Rosa26 locus with the loxP-stop-loxP sequence, which is commonly performed for conditional overexpression in mice [47,48]. This genetic recombination approach allows cells to inherit the recombined allele and continue to overexpress Sall4.
We chose to use TCre, which recombines broadly in meso-endoderm and neuromesodermal progenitors [21,34]. Sall4 is broadly expressed in gastrulating embryos, and as discussed above, conditional deletion of Sall4 in various types of progenitor cells demonstrated that Sall4 is required in embryo development. Moreover, Sall4 expression is rapidly downregulated from the limb bud mesenchyme, somites, and other areas of the embryo [14]. However, Sall4 overexpression did not cause significant developmental defects, and the overall morphology of these embryos and neonates is comparable to wild-type embryos or neonates. The lack of defects was surprising to us, given the conditional loss of Sall4 causes various defects during embryonic development. However, a similar observation was also reported in Xenopus, in which Sall4 was overexpressed by injecting mRNA into animal-dorsal cells of 4-cell stage embryos [45]. Compared to experiments by the same group, where Sall4 loss-of-function by morpholino injection resulted in a change in expression patterns of several neural markers, Sall4 overexpression did not affect the expression pattern of marker genes in early stage Xenopus embryos. In our study, while the overall morphology was not changed by overexpression of Sall4 in the T-lineage, we observed a slight reduction of limb long bone length. The reduction was very subtle, although statistically significant. Based on the comparison of bone length, we speculate that harboring the Sall4 expression cassette, although its expression is silenced, might have a subtle effect on skeletal growth.
Studies in non-mammalian animals suggested that Sall4 is involved in limb regeneration. Sall4 is upregulated during limb regeneration in Xenopus [23,24] and axolotls [25]. It has been hypothesized that Sall4 keeps cells in an undifferentiated state in the blastema during the early stages of blastema formation [23][24][25]. Our study showed that Sall4 is expressed in the digit tips of mouse fetuses and neonates. Mouse digit tips can regenerate after amputation at the terminal phalange level where the nail bed is associated [27]. The expression pattern is consistent with the postulated role of Sall4 in regeneration. However, our study showed that Sall4 overexpression does not affect digit tip regeneration. Overexpression of Sall4 did not interfere with the normal regenerative ability of the digit tip, when amputated at the terminal phalange. Moreover, Sall4 overexpression did not provide a regenerative ability, when amputated at the distal part of the second phalange level. One possible interpretation of our results is that Sall4 alone cannot provide limb regenerative ability. Sall4 may require highly regenerative cellular or tissue context to contribute to limb regeneration. Loss of function experiments in Xenopus or axolotls would provide insights into Sall4 function in limb regeneration.
A lack of evident phenotype after Sall4 overexpression suggests that SALL4 requires molecular partners in the experimental setting examined in this study. For example, SALL4 interacts with PLZF and DMRT1 in undifferentiated spermatogonial progenitor cells by coimmunoprecipitation experiments [17,49]. Our recent study also suggests that SALL4 functions, at least in part, depend on molecular partners. More specifically, SALL4 ChIP-seq in mouse ES cells [12] and in mesoderm tissues from the posterior part of E9.5 embryos exhibited significant difference in SALL4-enriched sequences [21]. Moreover, transcription factor motifs in the SALL4-enriched sequences are also distinct between ES cells and the embryonic tissue. These reports suggest that, in addition to directly binding to DNA [11], SALL4 also acts in a protein complex. In the latter case, Sall4 function may require cell or tissue-specific interacting partners.
Overall, our study in developing embryos and the neonatal digit showed that overexpression of Sall4 does not alter the developmental and regenerative processes that we examined. These results suggest that Sall4 acts as a permissive factor rather than an instructive factor in these developmental and regeneration contexts.
Although we did not observe significant effects by overexpressing Sall4 in the Brachyury lineage, our Sall4 overexpression model may be used in different contexts to gain insights into Sall4 functions in the future. It is suggested that Sall4 blocks differentiation of mouse ES cells [11,12]. It will be interesting to interrogate whether Sall4 overexpression can alter developmental processes that are not examined in this study, such as neural development. It will also be of interest to test whether Sall4 overexpression can alter regenerative processes that are not examined in this study, as well as cancer cell phenotypes.